PROJECT SUMMARY Although significant portions of the US population are older, overweight, obese, or living with conditions that involve increased deposition of adipose tissue in muscle, little is known about the potential mechanical effects of fat deposition on skeletal muscle function. Emerging evidence suggests that poor force and power production and transmission can occur in single muscle fibers and whole muscle in obese or older individuals. The potential negative consequences of the sequelae due to greater fat infiltration in muscle include weakness, increased risks for sedentary behavior, accelerated metabolic abnormalities and a general loss of mobility and independence. In this integrative and translational project, we will compare muscle force and power, and the amount and distribution of adipose tissue, from the molecular to whole-muscle level in the knee extensor muscles of healthy, sedentary: young (25-45 yr) low-fat, young high-fat, and older (65-75 yr) high-fat adults (8 men, 8 women per group). Noninvasive magnetic resonance imaging (MRI) and spectroscopy (MRS) techniques will be used to quantify key aspects of whole-muscle fat deposition and its effects on muscle architecture in vivo. We will also quantify the effects of adipose tissue on single muscle fiber function and address the potential molecular mechanisms of muscle dysfunction in subsets of 10 (5 men, 5 women) young low-fat, young high-fat and older high-fat adults who will undergo vastus lateralis muscle biopsies. In Aim 1, we will compare whole-muscle and single-fiber force- and power-velocity relationships in our study groups, as well as myofilament mechanics and myosin-actin cross-bridge kinetics, to quantify the extent to which these fundamental characteristics of muscle are altered with fat infiltration and/or older age. In Aim 2, we will use T1-weighted, 6-point Dixon, and diffusion tensor MRI to quantify the amount, location and effects on muscle fiber tracks of fat deposits. We will also measure intramyocellular lipid content in whole muscle and single fibers, using 1H MRS and oil red-O immuno-histochemistry, respectively. In Aim 3, we will evaluate whether the architectural disruptions due to adipose tissue deposition in muscle are associated with lower whole- muscle specific force and power, thus providing evidence of the pathological consequences of fat infiltration in otherwise healthy young and older adults. The results of this project will 1) develop critical new information that will address a significant knowledge gap in this understudied area of research, 2) provide original data to be used to formulate novel hypotheses and refine our approach to the important question of adipose-related muscle dysfunction in young and old, and 3) establish a unique translational line of investigation by an experienced research team. These objectives are highly relevant to the missions of NIH, NIAMS and NIA, and constitute an innovative project with the potential for rapidly generating transformative new knowledge. As numerous diseases and disorders, as well as aging, are associated with increased fat deposition in muscle tissue, the project has the potential to benefit the health of a significant portion of the U.S. population.